结直肠癌（CRC）是世界范围内的死亡首要原因。SRY转录因子9（SOX9）参与正常组织的器官发生和细胞分化，但也与癌变发展有关。癌细胞干细胞（CSCs）是存在于实体肿瘤中的一小部分细胞，它们会导致肿瘤异质性的增加、转移、耐药性和复发。CSCs具有自我更新和分化等特性，这些特性可以受到许多因素的调节。目前，SOX9在干细胞表型维持中的作用尚未完全阐明，因此，在本研究中，我们评估了SOX9缺失对CRC细胞干细胞表型的影响。我们通过敲除（KO）HCT116未分化CRC细胞系中的SOX9并利用球体形成试验、分化试验和免疫表型鉴定评估其干细胞特性。SOX9-KO影响了HCT116细胞的上皮形态和其干细胞特性，如增加了SOX2作为代偿性机制诱导SOX9表达的多能性特征标记，增加了KLF4作为分化特征标记，以及抑制了干细胞标记物CD44和CD73。此外，SOX9-KO细胞获得了上皮间质转化（EMT）表型，CDH2显著上调。此外，我们的结果显示，SOX9-KO细胞在第一和第二球形成中具有显著影响，即SOX9-KO细胞形成大尺寸耐药球的能力较差。然而，在分化试验中，CSCs表面标记物并未受到影响。总的来说，我们的研究结果提供了证据表明SOX9促进了CRC-CSCs的干细胞特性的维持。2023年《胃肠肿瘤学杂志》。版权所有。

Colorectal cancer (CRC) is a leading cause of death worldwide. SRY-box transcription factor 9 (SOX9) participates in organogenesis and cell differentiation in normal tissues but has been involved in carcinogenesis development. Cancer stem cells (CSCs) are a small population of cells present in solid tumors that contribute to increased tumor heterogeneity, metastasis, chemoresistance, and relapse. CSCs have properties such as self-renewal and differentiation, which can be modulated by many factors. Currently, the role of SOX9 in the maintenance of the stem phenotype has not been well elucidated, thus, in this work we evaluated the effect of the absence of SOX9 in the stem phenotype of CRC cells.We knockout (KO) SOX9 in the undifferentiated CRC cell line HCT116 and evaluated their stemness properties using sphere formation assay, differentiation assay, and immunophenotyping.SOX9-KO affected the epithelial morphology of HCT116 cells and stemness characteristics such as its pluripotency signature with the increase of SOX2 as a compensatory mechanism to induce SOX9 expression, the increase of KLF4 as a differentiation feature, as well as the inhibition of the stem cell markers CD44 and CD73. In addition, SOX9-KO cells gain the epithelial-mesenchymal transition (EMT) phenotype with a significant upregulation of CDH2. Furthermore, our results showed a remarkable effect on first- and second-sphere formation, being SOX9-KO cells less capable of forming high-size-resistant spheres. Nevertheless, CSCs surface markers were not affected during the differentiation assay.Collectively, our findings supply evidence that SOX9 promotes the maintenance of stemness properties in CRC-CSCs.2023 Journal of Gastrointestinal Oncology. All rights reserved.